Article | Published:

Single-cell transcriptome analysis reveals coordinated ectopic gene-expression patterns in medullary thymic epithelial cells

Nature Immunology 16, 933–941 (2015) | Download Citation

Subjects

Abstract

Expression of tissue-restricted self antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for the induction of self-tolerance and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and is coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA sequencing and obtained evidence of numerous recurring TRA–co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process that might involve local remodeling of chromatin and thus ensures a comprehensive representation of the immunological self.

  • Subscribe to Nature Immunology for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    et al. Projection of an immunological self shadow within the thymus by the aire protein. Science 298, 1395–1401 (2002).

  2. 2.

    & Lessons on immune tolerance from the monogenic disease APS1. Curr. Opin. Genet. Dev. 17, 193–200 (2007).

  3. 3.

    , & Central tolerance: learning self-control in the thymus. Nat. Rev. Immunol. 5, 772–782 (2005).

  4. 4.

    , , & Positive and negative selection of the T cell repertoire: what thymocytes see (and don′t see). Nat. Rev. Immunol. 14, 377–391 (2014).

  5. 5.

    , , & Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat. Immunol. 2, 1032–1039 (2001).

  6. 6.

    & A central role for central tolerance. Annu. Rev. Immunol. 24, 571–606 (2006).

  7. 7.

    et al. Distinct contributions of Aire and antigen-presenting-cell subsets to the generation of self-tolerance in the thymus. Immunity 41, 414–426 (2014).

  8. 8.

    , , , & Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance. Science 348, 589–594 (2015).

  9. 9.

    et al. Aire-dependent thymic development of tumor-associated regulatory T cells. Science 339, 1219–1224 (2013).

  10. 10.

    , , , & Promiscuous gene expression patterns in single medullary thymic epithelial cells argue for a stochastic mechanism. Proc. Natl. Acad. Sci. USA 105, 657–662 (2008).

  11. 11.

    et al. Expression of tumor-associated differentiation antigens, MUC1 glycoforms and CEA, in human thymic epithelial cells: implications for self-tolerance and tumor therapy. Cancer Res. 67, 3919–3926 (2007).

  12. 12.

    et al. Overlapping gene coexpression patterns in human medullary thymic epithelial cells generate self-antigen diversity. Proc. Natl. Acad. Sci. USA 110, E3497–E3505 (2013).

  13. 13.

    & Aire. Annu. Rev. Immunol. 27, 287–312 (2009).

  14. 14.

    , , & Aire's partners in the molecular control of immunological tolerance. Cell 140, 123–135 (2010).

  15. 15.

    et al. Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels. J. Exp. Med. 202, 33–45 (2005).

  16. 16.

    et al. Aire employs a histone-binding module to mediate immunological tolerance, linking chromatin regulation with organ-specific autoimmunity. Proc. Natl. Acad. Sci. USA 105, 15878–15883 (2008).

  17. 17.

    et al. The autoimmune regulator PHD finger binds to non-methylated histone H3K4 to activate gene expression. EMBO Rep. 9, 370–376 (2008).

  18. 18.

    et al. The transcriptional regulator Aire coopts the repressive ATF7ip-MBD1 complex for the induction of immunotolerance. Nat. Immunol. 15, 258–265 (2014).

  19. 19.

    et al. Population and single cell genomics reveal the Aire-dependency, relief from Polycomb silencing and distribution of self-antigen expression in thymic epithelia. Genome Res. 24, 1918–1931 (2014).

  20. 20.

    et al. Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells. Proc. Natl. Acad. Sci. USA 109, 535–540 (2012).

  21. 21.

    , , & Ectopic expression of peripheral-tissue antigens in the thymic epithelium: probabilistic, monoallelic, misinitiated. Proc. Natl. Acad. Sci. USA 105, 15854–15859 (2008).

  22. 22.

    et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat. Methods 10, 1096–1098 (2013).

  23. 23.

    et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).

  24. 24.

    , , , , & Transcriptome sequencing of neonatal thymic epithelial cells. Sci. Rep. 3, 1860 (2013).

  25. 25.

    et al. Accounting for technical noise in single-cell RNA-seq experiments. Nat. Methods 10, 1093–1095 (2013).

  26. 26.

    et al. A promoter-level mammalian expression atlas. Nature 507, 462–470 (2014).

  27. 27.

    et al. Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells. Nat. Biotechnol. 33, 155–160 (2015).

  28. 28.

    et al. Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages. Nat. Cell Biol. 16, 27–37 (2014).

  29. 29.

    , , & Medullary epithelial cells of the human thymus express a highly diverse selection of tissue-specific genes colocalized in chromosomal clusters. J. Exp. Med. 199, 155–166 (2004).

  30. 30.

    et al. Chromosomal clustering of genes controlled by the aire transcription factor. Proc. Natl. Acad. Sci. USA 102, 7233–7238 (2005).

  31. 31.

    , , , & Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

  32. 32.

    et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777–782 (2012).

  33. 33.

    et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat. Protoc. 5, 516–535 (2010).

  34. 34.

    et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat. Methods 6, 377–382 (2009).

  35. 35.

    et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res. 21, 1160–1167 (2011).

  36. 36.

    et al. Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing. Nat. Protoc. 7, 813–828 (2012).

  37. 37.

    et al. The impact of negative selection on thymocyte migration in the medulla. Nat. Immunol. 10, 823–830 (2009).

  38. 38.

    et al. Misinitiation of intrathymic MART-1 transcription and biased TCR usage explain the high frequency of MART-1-specific T cells. Eur. J. Immunol. 44, 2811–2821 (2014).

  39. 39.

    , , , & Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells. Nat. Med. 6, 56–61 (2000).

  40. 40.

    et al. Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat. Genet. 42, 53–61 (2010).

  41. 41.

    et al. Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells. Proc. Natl. Acad. Sci. USA 107, 19426–19431 (2010).

  42. 42.

    et al. Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532–538 (2006).

  43. 43.

    et al. Regional activation of the cancer genome by long-range epigenetic remodeling. Cancer Cell 23, 9–22 (2013).

  44. 44.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  45. 45.

    et al. Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells. J. Immunol. 194, 921–928 (2015).

  46. 46.

    , , & Epithelial heterogeneity in the murine thymus: a cell surface glycoprotein expressed by subcapsular and medullary epithelium. J. Histochem. Cytochem. 39, 645–653 (1991).

  47. 47.

    , , & Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells. J. Histochem. Cytochem. 36, 1511–1517 (1988).

  48. 48.

    & Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

Download references

Acknowledgements

We thank K. Hexel and S. Schmitt for single-cell sorting; S. Egle for technical help; C. Sebening and T. Loukanov (University of Heidelberg) for human thymic tissue; The Genomics Core Facility of the European Molecular Biology Laboratory for initial sequencing, and M. Miranda and E. Hopmans for support during subsequent sequencing at the Stanford Genome Technology Center; J. Buenrostro and C. Chabbert for discussions about ATAC-seq experiments and data, respectively; C. Michel and S. Anders for advice and comments on the manuscript; W. Wei and M. Sikora for help with data transfer; and The Central Animal Facility (German Cancer Research Center) for animal care. Supported by the European Union 7th Framework Programme (Health) via Project Radiant (W.H. and A.R.), The Helmholtz Center (K.R.), the Sonderforschungsbereich (DFG 938 to S.P.), the European Research Council (ERC-2012-AdG to B.K.) and the US National Institutes of Health (P01 HG000205 and R01 GM068717 to P.B., M.N. and L.M.S.).

Author information

    • Philip Brennecke
    • , Alejandro Reyes
    • , Sheena Pinto
    •  & Kristin Rattay

    These authors contributed equally to this work.

    • Wolfgang Huber
    • , Bruno Kyewski
    •  & Lars M Steinmetz

    These authors jointly directed this work.

Affiliations

  1. Department of Genetics, Stanford University, School of Medicine, California, USA.

    • Philip Brennecke
    • , Michelle Nguyen
    •  & Lars M Steinmetz

  2. Stanford Genome Technology Center, Stanford University, California, USA.

    • Philip Brennecke
    • , Michelle Nguyen
    •  & Lars M Steinmetz

  3. European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.

    • Alejandro Reyes
    • , Wolfgang Huber
    •  & Lars M Steinmetz

  4. Division of Developmental Immunology, German Cancer Research Center, Heidelberg, Germany.

    • Sheena Pinto
    • , Kristin Rattay
    • , Rita Küchler
    •  & Bruno Kyewski

Authors

  1. Search for Philip Brennecke in:

  2. Search for Alejandro Reyes in:

  3. Search for Sheena Pinto in:

  4. Search for Kristin Rattay in:

  5. Search for Michelle Nguyen in:

  6. Search for Rita Küchler in:

  7. Search for Wolfgang Huber in:

  8. Search for Bruno Kyewski in:

  9. Search for Lars M Steinmetz in:

Contributions

P.B., S.P., B.K. and L.M.S. conceived of the project; P.B., S.P. and K.R. designed experiments; P.B. performed single-cell sequencing experiments, Klk5 single-cell quantitative PCR confirmation experiments and ATAC-seq experiments; S.P. helped with the ATAC-seq experiments; S.P. and K.R. performed experimental mTEC preparations and flow cytometry of single and bulk mTECs; A.R. and W.H. designed analysis strategy and analyzed the data; A.R. prepared the figures; P.B., A.R., S.P., K.R., W.H., B.K. and L.M.S. interpreted the data and wrote the manuscript; M.N. and R.K. provided technical assistance; and L.M.S., B.K. and W.H. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Wolfgang Huber or Bruno Kyewski or Lars M Steinmetz.

Integrated supplementary information

Supplementary figures

  1. 1.

    Histogram of the fraction of genes detected per mTEC that are classified as TRA-encoding genes.

  2. 2.

    Enrichment of TRA-encoding genes among highly variable genes.

  3. 3.

    Enrichment of genes in the Tspan8–co-expressed gene set among genes from cluster B resulting from the k-medoids clustering.

  4. 4.

    Confirmation of co-expressed gene sets by independent experimental approaches.

  5. 5.

    Enrichment of genes in the Klk5–co-expressed gene set among genes from cluster D resulting from the k-medoids clustering.

  6. 6.

    Expected versus observed genomic proximity of the 11 groups of co-expressed genes resulting from the k-medoids clustering.

  7. 7.

    Karyogram of the genomic position of the 11 groups of co-expressed genes resulting from the k-medoids clustering.

  8. 8.

    Examples of co-expressed genomic loci and their locations in the genome.

  9. 9.

    Fraction of cells with detection of expression of genes encoding proteases of the kallikrein family.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9

  2. 2.

    Supplementary Code 1

    Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

Excel files

  1. 1.

    Supplementary Table 1

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ni.3246

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.